Powassan virus (POWV), a North American tick-borne flavivirus (Flaviridae;flavivirus), is related to Old World tick-borne encephalitis (TBE) viruses found in Asia and Europe. Phylogenetic analyses of isolates indicated two lineages of POWV, prototype POWV transmitted predominantly by Ixodes cookei, and an antigenic variant, deer tick virus (DTV), transmitted by Ixodes scapularis. Although infections with POWV and DTV have been detected in various mammals, which of these species act as important reservoirs is unknown. Increased human surveillance following the introduction of West Nile virus into North America, and improved diagnostic reagents have demonstrated increased prevalence in North America of serious infection with POWV/DTV in humans. The overall goals of this study are to elucidate the dynamics of transmission of this emerging flavivirus and to provide the foundation for understanding risk. Our hypothesis is that POWV, and especially its antigenic variant, DTV, represent an emerging tick-borne pathogen that presents an increasing risk to humans as a consequence of burgeoning tick populations in the northeastern US. Integrated field and lab studies will (1) determine the environmental burden of POWV in ticks and their vertebrate hosts at enzootic foci;(2) determine which host(s) is responsible for infecting tick vectors;(3) further characterize the biology and ecology of POWV (and DTV);and (4) assess the role of birds as potential sources of virus for questing ticks. This pilot study will provide the foundation and guidance for future exhaustive efforts to understand spatial/temporal genetic differences between Lineage 1 (prototype POWV) and Lineage 2 (DTV) Powassan virus, geographic scope of transmission activity, role of co-feeding, and robust vector and host competence experiments. PUBLIC HEALTH RELEVANCE: Powassan encephalitis, caused by Powassan virus and its antigenic variant, deer tick virus, is of significant public health importance because of its serious nature in spite of its rarity. It is associated with significant long- term morbidity including severe neurologic sequelae with a case-fatality rate of 10%-15%. Because there is no vaccine or specific therapy for Powassan encephalitis, it is important to understand the ecology of the causative agent and how this virus interacts with its tick vectors and vertebrate hosts.